Variation in the Slimy Salamander (Plethodon spp.) Skin and Gut-Microbial Assemblages Is Explained by Geographic Distance and Host Affinity.

A multicellular host and its microbial communities are recognized as a metaorganism-a composite unit of evolution. Microbial communities have a variety of positive and negative effects on the host life history, ecology, and evolution. This study used high-throughput amplicon sequencing to characterize the complete skin and gut microbial communities, including both bacteria and fungi, of a terrestrial salamander, Plethodon glutinosus (Family Plethodontidae). We assessed salamander populations, representing nine mitochondrial haplotypes ('clades'), for differences in microbial assemblages across 13 geographic locations in the Southeastern United States. We hypothesized that microbial assemblages were structured by both host factors and geographic distance. We found a strong correlation between all microbial assemblages at close geographic distances, whereas, as spatial distance increases, the patterns became increasingly discriminate. Network analyses revealed that gut-bacterial communities have the highest degree of connectedness across geographic space. Host salamander clade was explanatory of skin-bacterial and gut-fungal assemblages but not gut-bacterial assemblages, unless the latter were analyzed within a phylogenetic context. We also inferred the function of gut-fungal assemblages to understand how an understudied component of the gut microbiome may influence salamander life history. We concluded that dispersal limitation may in part describe patterns in microbial assemblages across space and also that the salamander host may select for skin and gut communities that are maintained over time in closely related salamander populations.

Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards).

Presence of an invasive species reverses latitudinal clines of multiple traits in a native species.

Understanding the processes driving formation and maintenance of latitudinal clines has become increasingly important in light of accelerating global change. Many studies have focused on the role of abiotic factors, especially temperature, in generating clines, but biotic factors, including the introduction of non-native species, may also drive clinal variation. We assessed the impact of invasion by predatory fire ants on latitudinal clines in multiple fitness-relevant traits-morphology, physiological stress responsiveness, and antipredator behavior-in a native fence lizard. In areas invaded by fire ants, a latitudinal cline in morphology is opposite both the cline found in museum specimens from historical populations across the species' full latitudinal range and that found in current populations uninvaded by fire ants. Similarly, clines in stress-relevant hormone response to a stressor and in antipredator behavior differ significantly between the portions of the fence lizard range invaded and uninvaded by fire ants. Changes in these traits within fire ant-invaded areas are adaptive and together support increased and more effective antipredator behavior that allows escape from attacks by this invasive predator. However, these changes may mismatch lizards to the environments under which they historically evolved. This research shows that novel biotic pressures can alter latitudinal clines in multiple traits within a single species on ecological timescales. As global change intensifies, a greater understanding of novel abiotic and biotic pressures and how affected organisms adapt to them across space and time will be central to predicting and managing our changing environment.

Stressed snakes strike first: Hormone levels and defensive behavior in free ranging cottonmouths (Agkistrodon piscivorus).

Stress is believed to be an important factor mediating animal behavior. Here we explore the relationship between concentrations of a stress hormone and defensive behavior of a snake. The cottonmouth (Agkistrodon piscivorus) is an abundant, large-bodied pitviper that is well known for its intense defensive behaviors. The defensive behaviors and hormonal ecology of cottonmouths have been studied extensively, but the interaction between these is not well understood. We conducted field trials, recording the snake's behavior and obtaining blood samples to quantify plasma CORT concentrations, both upon first encountering a snake and after a 30min standardized confinement stressor. We found that snakes with elevated levels of baseline CORT at first encounter were more likely to strike than exhibit a threat display when approached in the field. However, this behavior was not related to the magnitude of the snake's CORT increase following confinement, suggesting that more stress-prone snakes are not more defensive. Post-stressor antipredator behavior was also not related to any of our CORT measures. This study suggests that baseline CORT levels can be important correlates of defensive behavior. If this is a causative relationship, environmental challenges that increase baseline stress levels of populations may elevate cottonmouth defensive behavior. This would increase costs associated with defensive behavior (energetic, lost opportunity, etc.) and have important consequences for animal-human interactions.

Corticosterone-immune interactions during captive stress in invading Australian cane toads (Rhinella marina).

Vertebrates cope with physiological challenges using two major mechanisms: the immune system and the hypothalamic pituitary-adrenal axis (e.g., the glucocorticoid stress response). Because the two systems are tightly integrated, we need simultaneous studies of both systems, in a range of species, to understand how vertebrates respond to novel challenges. To clarify how glucocorticoids modulate the amphibian immune system, we measured three immune parameters and plasma corticosterone (CORT), before and after inflicting a stressor (capture and captive confinement) on introduced cane toads (Rhinella marina) near their invasion front in Australia. Stress increased CORT levels, decreased complement lysis capacity, increased leukocyte oxidative burst, and did not change heterologous erythrocyte agglutination. The strength of the CORT response was positively correlated with leukocyte oxidative burst, and morphological features associated with invasiveness in cane toads (relative leg length) were correlated with stress responsiveness. No immune parameter that we measured was affected by a toad's infection by a parasitic nematode (Rhabdias pseudosphaerocephala), but the CORT response was muted in infected versus uninfected toads. These results illustrate the complex immune-stress interactions in wild populations of a non-traditional model vertebrate species, and describe immune adaptations of an important invasive species.

The impacts of invaders: basal and acute stress glucocorticoid profiles and immune function in native lizards threatened by invasive ants.

As anthropogenic stressors increase exponentially in the coming decades, native vertebrates will likely face increasing threats from these novel challenges. The success or failure of the primary physiological mediator of these stressors--the HPA axis--will likely involve numerous and chaotic outcomes. Among the most challenging of these new threats are invasive species. These have the capacity to simultaneously challenge the HPA axis and the immune system as they are often associated with, or the cause of, emerging infectious diseases, and energetic tradeoffs with the HPA response can have immunosuppressive effects. To determine the effects of invasive species on the vertebrate GC response to a novel stressor, and on immunity, we examined the effects of invasive fire ants on native lizards, comparing lizards from sites with long histories with fire ants to those outside the invasion zone. We demonstrated higher baseline and acute stress (captive restraint) CORT levels in lizards from within fire ant invaded areas; females are more strongly affected than males, suggesting context-specific effects of invasion. We found no effect of fire ant invasion on the immune parameters we measured (complement bacterial lysis and antibody hemagglutination) with the exception of ectoparasite infestation. Mites were far less prevalent on lizards within fire ant invaded sites, suggesting fire ants may actually benefit lizards in this regard. This study suggests that invasive species may impose physiological stress on native vertebrates, but that the consequences of this stress may be complicated and unpredictable.

Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates.

Phylogeography of the Brownback Salamander reveals patterns of local endemism in Southern Appalachian springs.

The Appalachian Mountains of eastern North America are characterized by high faunal diversity and many endemic species, especially in the unglaciated southern latitudes where lineages have been accumulating for tens of millions of years. The Brownback Salamander, Eurycea aquatica, is an enigmatic species that dwells in isolated springs in southeastern North America. Eurycea aquatica have often been dismissed as simply robust spring-adapted ecomorphs of the widespread and more gracile species Eurycea cirrigera. We sequenced the mitochondrial gene encoding NADH dehydrogenase subunit-2 (ND2; 753 bp) and the nuclear recombination activating gene-1 (Rag1; 1201 bp) for E. aquatica (ND2 n = 72; Rag1 n = 17) from across their presumed distribution and compared them to E. cirrigera (ND2 n = 23; Rag1 n = 10) from nearby populations. Using phylogenetic and morphological analyses we explicitly test if E. aquatica in the Southern Appalachians is simply a local spring-adapted ecomorph of E. cirrigera or a single lineage that resulted from fragmentation of (or dispersal to) spring habitats. We found that E. aquatica from isolated springs form a well-supported monophyletic group that is nested among E. cirrigera, E. wilderae, and E. junaluska. Furthermore, we uncovered three very divergent lineages of E. aquatica that we estimate have been isolated from each another since the early Pliocene to late Miocene (2.5-6.1 Myr) and may each represent distinct species. The distribution of these lineages is coincident with the distribution of other endemic spring-dwelling vertebrates, and suggests that this region may be a relictual habitat for an unexpected diversity of unrecognized endemics.

The reproductive biology of male cottonmouths (Agkistrodon piscivorus): do plasma steroid hormones predict the mating season?

To better understand the proximate causation of the two major types of mating seasons described for North American pitvipers, we conducted a field study of the cottonmouth (Agkistrodon piscivorus) in Georgia from September 2003 to May 2005 that included an extensive observational regime and collection of tissues for behavioral, anatomical, histological, and hormone analysis. Enzyme immunoassays (EIA) of plasma samples and standard histological procedures were conducted on reproductive tissues. Evidence from the annual testosterone (T) and sexual segment of the kidney (SSK) cycle and their relationship to the spermatogenic cycle provide correlative evidence of a unimodal mating pattern in this species of pitviper, as these variables consistently predict the mating season in all snake species previously examined under natural conditions. In most reptiles studied to date, high plasma levels of T and corticosterone (CORT) coincide during the mating period, making the cottonmouth an exception to this trend; we suggest two possible explanations for increased CORT during spring (regulation of a spring basking period), and decreased CORT during summer (avoiding reproductive behavioral inhibition), in this species.

Description of an extant salamander from the Gulf Coastal Plain of North America: The Reticulated Siren, Siren reticulata.

The salamander family Sirenidae is represented by four extant species that are restricted to North America. Sirens are abundant throughout the southern United States and are among the world's largest amphibians, yet the biology, ecology, and phylogeography of this group is poorly-known. In this study we use morphological and genetic evidence to describe a previously unrecognized species from southern Alabama and the Florida panhandle. We name this species the Reticulated Siren, Siren reticulata. Future studies will enable more precise phylogenetic information about S. reticulata and will almost surely reveal additional undescribed species within the family.

A salamander's top down effect on fungal communities in a detritivore ecosystem.

The soil decomposer community is a primary driver of carbon cycling in forest ecosystems. Understanding the processes that structure this community is critical to our understanding of the global carbon cycle. In North American forests, soil fungal communities are regulated by grazing soil invertebrates, which are in turn controlled by the predatory red-backed salamander (Plethodon cinereus). The presence of these soil invertebrate taxa is known to exert direct top-down control via selective grazing on saprotrophic fungi, with direct consequences for biogeochemical cycling in soil. We investigated whether the removal of P. cinereus would relieve top-down control on decomposer fungal communities in a tri-trophic mesocosm study. Fungal communities were characterized using metabarcoding and high-throughput DNA sequencing. The ß-diversity of fungal communities differed between salamander presence and absence treatments with a strong effect on saprotrophic fungal communities. We concluded that P. cinereus, a mesopredator in the detritivore food chain, exerts a prominent control on the composition and functional diversity of fungal communities in soil through a multi-trophic top-down process. Given their capacity to govern the compositions of soil invertebrates, the activity of these amphibians may be important for regulating ecosystem function and nutrient cycling in temperate forest systems.

Thermal performance and acclimatization of a component of snake (Agkistrodon piscivorus) innate immunity.

Complement-an immune protein cascade involved in pathogen lysis-was discovered as a temperature-labile component of vertebrate plasma, yet since that time the thermal performance of complement has not received much attention from a comparative or ecological perspective. We investigated two thermal hypotheses involving the complement system of the cottonmouth snake (Agkistrodon piscivorus). We tested whether complement performance would conform to optimal thermal reaction norms commonly observed in ectotherm ecophysiological studies, predicting that complement efficiency would be maximal at or near the cottonmouth's field body temperatures. We also tested thermal acclimatization of complement performance, by comparing temperature/performance curves from samples collected in three different seasons. Complement efficiency exhibited the same significant positive correlation with temperature in all three seasons. This seasonally invariable temperature-performance relationship may allow easy acquisition of behavioral fever, as well as trade-offs between immune performance and energy balance, ultimately endowing snakes with immunological flexibility not available to endotherms.

Are Invasive Species Stressful? The Glucocorticoid Profile of Native Lizards Exposed to Invasive Fire Ants Depends on the Context.

Invasive species represent a substantial threat to native species worldwide. Research on the impacts of invasive species on wild living vertebrates has focused primarily on population-level effects. The sublethal, individual-level effects of invaders may be equally important but are poorly understood. We investigated the effects of invasive fire ants (Solenopsis invicta) on the physiological stress response of a native lizard (Sceloporus undulatus) within two experimental contexts: directly exposing lizards to a fire ant attack and housing lizards with fire ants in seminatural field enclosures. Lizards directly exposed to brief attack by fire ants had elevated concentrations of the stress hormone corticosterone (CORT), suggesting that these encounters can be physiologically stressful. However, lizards exposed for longer periods to fire ants in field enclosures had lower concentrations of CORT. This may indicate that the combined effects of confinement and fire ant exposure have pushed lizards into allostatic overload. However, lizards from fire ant enclosures appeared to have intact negative feedback controls of the stress response, evidenced by functioning adrenocorticotropic hormone responsiveness and lack of suppression of innate immunity (plasma bactericidal capacity). We review previous studies examining the stress response of wild vertebrates to various anthropogenic stressors and discuss how these-in combination with our results-underscore the importance of considering context (the length, frequency, magnitude, and types of threat) when assessing these impacts.

Variation in mating systems of salamanders: mate guarding or territoriality?

Two of the most common mating tactics in vertebrates are mate guarding and territoriality, yet much of the research on these strategies has focused on mating systems in birds, despite novel insights gained from studying less traditional systems. North American stream salamanders that comprise the Eurycea bislineata complex represent an excellent nontraditional system for comparing mating strategies because these species exhibit a continuum of male morphologies, diverse habitat associations, and various potential mating strategies. We studied two species within this complex that exhibit the extremes of this continuum, Eurycea aquatica (robust morph) and Eurycea cirrigera (slender morph). The larger head in males of E. aquatica is due to larger musculature around the jaw and may be associated with aggressive behavior. Therefore, we hypothesized that the robust morphology exhibited by males of E. aquatica provides benefits during either territorial defense or mate defense and that males of E. cirrigera would not exhibit aggression in either scenario. We found that neither species exhibited aggressive behavior to defend a territory. However, in the presence of a female, males of E. aquatica were significantly more aggressive toward intruding males than were males of E. cirrigera. Therefore, mate-guarding behavior occurs in E. aquatica, and the enlarged head of males likely aids in deterring rivals. This is the first demonstration of mate-guarding behavior in a plethodontid, the most speciose family of salamanders.

A metagenomics-based approach to the top-down effect on the detritivore food web: a salamanders influence on fungal communities within a deciduous forest.

The flow of energy within an ecosystem can be considered either top-down, where predators influence consumers, or bottom-up, where producers influence consumers. Plethodon cinereus (Red-backed Salamander) is a terrestrial keystone predator who feeds on invertebrates within the ecosystem. We investigated the impact of the removal of P. cinereus on the detritivore food web in an upland deciduous forest in northwest Ohio, U.S.A. A total of eight aluminum enclosures, each containing a single P. cinereus under a small log, were constructed in the deciduous forest. On Day 1 of the experiment, four salamanders were evicted from four of the eight enclosures. Organic matter and soil were collected from the center of each enclosure at Day 1 and Day 21. From each sample, DNA was extracted, fungal-specific amplification performed, and 454 pyrosequencing was used to sequence the nuclear ribosomal internal transcribed spacer (ITS2) region and partial ribosomal large subunit (LSU). Changes in overall fungal community composition or species diversity were not statistically significant between treatments. Statistically significant shifts in the most abundant taxonomic groups of fungi were documented in presence but not absence enclosures. We concluded that P. cinereus does not affect the overall composition or diversity of fungal communities, but does have an impact on specific groups of fungi. This study used a metagenomics-based approach to investigate a missing link among a keystone predator, P. cinereus, invertebrates, and fungal communities, all of which are critical in the detritivore food web.

Resting environments of some Costa Rican mosquitoes.

The resting sites of tropical American mosquitoes are poorly documented, and the few reports that do exist are largely from opportunistic collections. Since blood-engorged females (used in determining host associations) are more efficiently collected from resting sites than attractive traps, information on resting site utilization has practical value. To investigate differences in the resting sites utilized by tropical mosquitoes, we collected and identified female mosquitoes from one man-made (resting shelter) and three natural (buttress tree roots, hollow trees, and understory vegetation) resting environments at a tropical dry forest location in western Costa Rica. All of the most common species collected demonstrated associations with one or more resting environments. Females of five species (blood-engorged Anopheles albimanus, Uranotaenia apicalis, Uranotaenia lowii, Uranotaenia orthodoxa, and blood-engorged Mansonia titillans) were collected in significantly greater numbers from understory vegetation than other resting environments. Culex erraticus and other members of the subgenus Melanoconion were encountered more often in resting shelters, hollow trees, and buttress roots, while Culex restrictor (blood-engorged) females were associated with hollow trees. Similarity indices indicate that buttress tree roots, hollow trees, and resting shelters are similar with respect to the mosquito communities that utilize them as resting sites, while understory vegetation has a resting fauna that is different than the other environments surveyed here. These results add to the body of information regarding resting sites utilized by tropical American mosquitoes.

Serosurveillance of eastern equine encephalitis virus in amphibians and reptiles from Alabama, USA.

Eastern equine encephalitis virus (EEEV) is among the most medically important arboviruses in North America, and studies suggest a role for amphibians and reptiles in its transmission cycle. Serum samples collected from 351 amphibians and reptiles (27 species) from Alabama, USA, were tested for the presence of antibodies against EEEV. Frogs, turtles, and lizards showed little or no seropositivity, and snakes had high seropositivity rates. Most seropositive species were preferred or abundant hosts of Culex spp. mosquitoes at Tuskegee National Forest, that target ectothermic hosts. The cottonmouth, the most abundant ectotherm sampled, displayed a high prevalence of seropositivity, indicating its possible role as an amplification and/or over-wintering reservoir for EEEV.

Detection of eastern equine encephalomyelitis virus RNA in North American snakes.

The role of non-avian vertebrates in the ecology of eastern equine encephalomyelitis virus (EEEV) is unresolved, but mounting evidence supports a potential role for snakes in the EEEV transmission cycle, especially as over-wintering hosts. To determine rates of exposure and infection, we examined serum samples from wild snakes at a focus of EEEV in Alabama for viral RNA using quantitative reverse transcription polymerase chain reaction. Two species of vipers, the copperhead (Agkistrodon contortrix) and the cottonmouth (Agkistrodon piscivorus), were found to be positive for EEEV RNA using this assay. Prevalence of EEEV RNA was more frequent in seropositive snakes than seronegative snakes. Positivity for the quantitative reverse transcription polymerase chain reaction in cottonmouths peaked in April and September. Body size and sex ratios were not significantly different between infected and uninfected snakes. These results support the hypothesis that snakes are involved in the ecology of EEEV in North America, possibly as over-wintering hosts for the virus.

Host reproductive phenology drives seasonal patterns of host use in mosquitoes.

Seasonal shifts in host use by mosquitoes from birds to mammals drive the timing and intensity of annual epidemics of mosquito-borne viruses, such as West Nile virus, in North America. The biological mechanism underlying these shifts has been a matter of debate, with hypotheses falling into two camps: (1) the shift is driven by changes in host abundance, or (2) the shift is driven by seasonal changes in the foraging behavior of mosquitoes. Here we explored the idea that seasonal changes in host use by mosquitoes are driven by temporal patterns of host reproduction. We investigated the relationship between seasonal patterns of host use by mosquitoes and host reproductive phenology by examining a seven-year dataset of blood meal identifications from a site in Tuskegee National Forest, Alabama USA and data on reproduction from the most commonly utilized endothermic (white-tailed deer, great blue heron, yellow-crowned night heron) and ectothermic (frogs) hosts. Our analysis revealed that feeding on each host peaked during periods of reproductive activity. Specifically, mosquitoes utilized herons in the spring and early summer, during periods of peak nest occupancy, whereas deer were fed upon most during the late summer and fall, the period corresponding to the peak in births for deer. For frogs, however, feeding on early- and late-season breeders paralleled peaks in male vocalization. We demonstrate for the first time that seasonal patterns of host use by mosquitoes track the reproductive phenology of the hosts. Peaks in relative mosquito feeding on each host during reproductive phases are likely the result of increased tolerance and decreased vigilance to attacking mosquitoes by nestlings and brooding adults (avian hosts), quiescent young (avian and mammalian hosts), and mate-seeking males (frogs).

Nestedness of ectoparasite-vertebrate host networks.

Determining the structure of ectoparasite-host networks will enable disease ecologists to better understand and predict the spread of vector-borne diseases. If these networks have consistent properties, then studying the structure of well-understood networks could lead to extrapolation of these properties to others, including those that support emerging pathogens. Borrowing a quantitative measure of network structure from studies of mutualistic relationships between plants and their pollinators, we analyzed 29 ectoparasite-vertebrate host networks--including three derived from molecular bloodmeal analysis of mosquito feeding patterns--using measures of nestedness to identify non-random interactions among species. We found significant nestedness in ectoparasite-vertebrate host lists for habitats ranging from tropical rainforests to polar environments. These networks showed non-random patterns of nesting, and did not differ significantly from published estimates of nestedness from mutualistic networks. Mutualistic and antagonistic networks appear to be organized similarly, with generalized ectoparasites interacting with hosts that attract many ectoparasites and more specialized ectoparasites usually interacting with these same "generalized" hosts. This finding has implications for understanding the network dynamics of vector-born pathogens. We suggest that nestedness (rather than random ectoparasite-host associations) can allow rapid transfer of pathogens throughout a network, and expand upon such concepts as the dilution effect, bridge vectors, and host switching in the context of nested ectoparasite-vertebrate host networks.